Modification of immune reactions by antigen-immunosuppressive agent conjugates. V. Studies on antigen-specific activity of 6-mercaptopurine bovine gamma globulin conjugates on the humoral immune response in rabbits]

Rabbits treated daily and for seven consecutive days with 6-mercaptopurine bovine gamma globulin conjugates (MPI--n-BGG; 'I'--characterizes the king of chemical binding and 'n' the number of coupled MP-residues per one mole BGG) show an altered immunological reactivity. A following intradermal immunization with BGG alum adsorbate results in a suppressed anti BGG antibody production on the third day after antigen application (antibody titer 1:320, antibody titer of control animals--pretreated with BGG and uncoupled 6-MP equals 1:5120). Already three days later the antibody titers of the test groups show a significant increase and are two dilution stages higher than the titers of the controls. A suppressive effect on the third day is induced by MPI--13-BGG, MPI--26-BGG, MPI--36-BGG and MPI--46-BGG; the later adjuvant effect can only be seen in the MPI--26-BGG, MPI--36-BGG and MPI--46-BGG but not in the MPI--13-BGG pretreated animal group. While the short time suppression was antigen specific--the humoral immune response against a second unrelated antigen was not reduced--the adjuvans effect was not antigen specific. A pretreatment with the substances mentioned above results in an increased anti BGG and anti HSA serum antibody level. Comparing investigations on the unspecific immunosuppression in rabbits by 6-mercaptopurine shows that application of 10 mg 6-MP/kg/day for seven days at first leads to a suppression but later on to an enhancement of antibody production. Application of 10 mg 6-MP/kg/day for 10 days results in a long lasting suppression without enhancement effect. As a reason for these differences the different catabolism of immunosuppressive agent and antigen is discussed. For the phenomena following antigen specific immunosuppression, similar mechanisms can be responsible.     

Modification of the immune reaction by antigen-immunosuppressive-agent conjugates. II. Immunogenicity of antigen-immunosuppressive-agent conjugates]

The maintenance of specific immunogenicity of carrier proteins is a necessary condition for the successful use of antigen-immunosuppressive agent-conjugates (AIC) for an antigen specific suppression of the immune response. The experimental results indicate that, in spite of the binding of 6-mercaptopurine (6-MP) and toluyl (T) to bovine gamma globulin (BGG) and human serum albumin (HSA), the carrier specific immunogenicity is not significantly altered. The intradermal application of 6-MP-BGG and T-BGG emulsified with complete Freund's adjuvant in guinea pigs results, in all cases, in a well detectable anti BGG hemagglutination and precipitation titer. This kind of immunization leads also to a formation of anti 6-mercaptopurine and anti new antigen determinants (NAD's) antibodies.     

Studies on the significance of antigen in the 6-mercapopurine-influenced humoral antibody response in guinea pigs]

The mechanism of suppression of humoral immune response to dinitrophenylated bovine gamma globulin (DNP23-BGG), human serum albumin (HSA), and trinitrophenylated sheep red blood cells (TNP-SRBC) by 6-mercaptopurine (6-MP) was studied in guinea pigs. Following the intradermal application of the antigens emulsified in complete (CFA) or incomplete Freund's adjuvant (IFA) each test animal was given 6-MP, 10 mg/kg/day for 7 days. This treatment resulted in a significant suppression of the anti BGG and anti SRBC agglutinating and complement binding antibody production. The latter was only significantly suppressed if the TNP-SRBC were applied together with CFA and not if TNP-SRBC were given in IFA. The anti DNP and anti HSA antibody formation was not influenced.     

Modification of the immune reaction by antigen-immunosuppressive-agent conjugates. I. Tentative hypothesis for the induction of antigen-specific suppression by antigen-immunosuppressive-agent conjugates]

The most important mechanisms for the specific depression of immune reactions--immuno-tolerance, enhancement, transfer of antibodies, drug induced tolerance, immunological suicide, application of antibody-toxin-complexes--are discussed with regard to their possible application in the clinical practice. A tentative hypothesis for induction of antigen specific suppression is proposed, basing on the use of antigen-immunosuppressive agent-conjugates (AIC). Antigen binding lymphocytes are supposed to bind the AIC and to pick them up through endocytosis. After breakdown of the AIC in the lymphoid cells the free immunosuppressive agent can become effective causing damage to the specific cell clones.     

Characterization and genetic control of the immune response to synthetic polypeptide antigens of defined geometry.

Both humoral and cell-mediated immune responses to the synthetic helical hapten-carrier conjugate poly-Glu-Tyr-Lys(TNP)-(Glu-Tyr-Ala)5 were found to be linked to the major histocompatibility locus in mice and guinea pigs. The responder mouse strains (H-2d haplotype) showed a primary IgM response with an IgG component appearing after the secondary immunization. The antibody response was accompanied by a positive DTH reaction in responder strains. Nonresponder mice (H-2b or H-2k haplotypes) showed neither IgM nor IgG antibodies and the DTH reaction was negative. Administration of the antigen as a complex with an immunogenic carrier was not effective in inducing a response in nonresponder mice. In guinea pig studies, it was found that strain 2 animals were able to mount an antibody response against the TNP-hapten and a DTH response against the polypeptide backbone. Strain 13 animals gave no anti-TNP antibodies at the lower dose levels and DTH activity was entirely negative for all doses of immunizing antigen. Replacement of the TNP hapten by the arsanilazo dipeptide derivative, BOC-gly-ARA-tyrosine, converted the nonresponder strain 13 guinea pigs into complete responders showing antibody and DTH reactions to both the hapten and the polypeptide backbone.     

Effects of cyclophosphamide on the expression and induction of delayed hypersensitivity.

Erythematous delayed reactions without induration, presumably assigned to Jones-Mote type, were characterized by the resistance to treatment with cyclophosphamide (CY) before elicitation or immunization in guinea pigs immunized with BGG in IFA or CFA. CY-treatment before elicitation converted delayed erythematous reactions from negative to positive at late intervals after immunization with BGG in IFA. Such a treatment augmented erythematous delayed reactions in animals immunized with BGG in CFA, but abolished induration at the reaction sites. CY-treatment before elicitation or immunization reduced the numbers of basophils at the reaction sites, although erythematous delayed reactions were augmented. Effector T cells responsible for delayed erythematous reaction without induration appear to persist for a long period of time after immunization in the presence of antibody production or tuberculin hypersensitivity and the expression of their function may be inhibited by suppressive mechanisms.     

Regulatory mechanisms of cutaneous delayed-type hypersensitivity. II. Suppression of cutaneous delayed-type hypersensitivity by macrophage disappearance reaction

Studies were performed on the behavior of cutaneous delayed-type hypersensitivity (DTH) in guinea pigs in which macrophage disappearance reaction (MDR) was induced. Guinea pigs were immunized with dinitrophenylated egg albumin (DNP-EA), followed by intraperitoneal (ip) injection of liquid paraffin in order to elicit peritoneal macrophages. Subsequently 20 micrograms of EA was injected into these animals and the animals were divided into two groups. One group of animals was sacrificed for estimation of MDR 6 hr after the subsequent ip injection. The other group received a skin test by EA at the time of the subsequent ip injection. The first group of animals sacrificed for estimation of MDR exhibited a marked reduction in the number of peritoneal macrophages. The second group of animals that received skin tests revealed suppressed skin reactions 24 hr after the subsequent ip injection. A similar experiment was performed using the guinea pigs doubly immunized with DNP-EA and dinitrophenylated bovine gamma-globulin (DNP-BGG). Induction of MDR was performed by ip injection of BGG and skin tests were done by both EA and BGG. As a result, suppression of not only BGG-induced skin reactions but also EA-induced skin reactions was observed in animals in which MDR had been induced by BGG. In addition, the guinea pigs in which MDR was induced showed hyporeactivity to phytohemagglutinin (PHA). Reactivity to skin reactive factor (SRF) was also suppressed in these animals. The culture supernatants of macrophages incubated with the MIF fraction in vitro showed the ability to suppress skin reactions of cutaneous DTH, PHA and SRF.     

Enhancement of macrophage bactericidal capacity by antigenically stimulated immune lymphocytes

The ability of antigenically stimulated immune lymphocytes to influence the bactericidal capacity of normal macrophages was studied in vitro. Purified lymphocytes were obtained from the lymph nodes and peritoneal exudates of guinea pigs immunized with bovine gamma globulin (BGG) and from control animals. Immune and control lymphocytes were added to normal macrophages and incubated overnight in the presence or absence of BGG. After washing, the macrophage monolayers were infected with Listeria monocytogenes; 4 hr later, the cells were lysed and the surviving intracellular bacteria quantitated. The macrophages which had been incubated with BGG-immune lymphocytes in the presence of BGG displayed a markedly enhanced listericidal capacity. In parallel experiments, these same antigen-stimulated lymphocytes were shown to inhibit the migration of normal macrophages. Lymphocytes derived from peritoneal exudates were more active than lymph node lymphocytes in both assays.     

Protection against experimental toxoplasmosis by adoptive immunotherapy

The role of humoral and cell-mediated immunity against toxoplasmosis in experimentally infected guinea pigs was examined by using a syngeneic passive transfer system. Serum or spleen and lymph node cells from guinea pigs immune to infection with the RH strain of Toxoplasma gondii conferred partial protection against symptomatic disease in recipient guinea pigs. This result was based on the reduced dissemination or growth of T. gondii parasites from the primary inoculation site to various selected organ sites of the recipients of immune serum or cells. Similar levels of partial protection against disseminated toxoplasmosis occurred in animals infused with cell suspensions enriched for immune T cells, whereas treatment of immune cells with a monoclonal anti-guinea pig T cell antibody plus complement abolished their ability to transfer resistance. These findings provide substantial direct evidence implicating both cellular and humoral components of the immune response as important effector mechanisms in host resistance to toxoplasmosis.     

Experimental cell-mediated interstitial nephritis induced with exogenous antigens.

The capacity of intrarenal injections of bovine gamma-globulin (BGG) to elicit hypersensitivity reactions was studied in guinea pigs immunized with DNP BGG or BGG immune complexes in CFA or in rats immunized with BGG in CFA. In guinea pigs it was found that heat-aggregated BGG elicited inflammatory reactions in the renal cortex, whereas soluble BGG did not. In rats only aggregated BGG was used, and this was found to be effective. The reactions were characterized by a predominantly mononuclear cell infiltrate. Transfer experiments were performed in rats and it was found that reactivity was transferrable with lymph node cells but not with serum.     

Adjuvant actions of polyclonal lymphocyte activators. II. Comparison and characterization of their actions in initiation and potentiation of immune responses to T-dependent and T-independent soluble antigens

Various polyclonal lymphocyte activators (PLA) such as capsular polysaccharide of Klebsiella pneumoniae (CPS-K), lipopolysaccharide of Escherichia coli (LPS), dextran sulfate (DS), concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM), and polyadenylic-polycytidylic acid (poly A:U) were compared in their effects on antibody response to T-dependent antigen (bovine gamma globulin (BGG) and dinitrophenylated (DNP)-BGG) and T-independent antigen (DNP-Ficoll) and on induction of tolerance to T-dependent antigen. All of these PLA acted more or less to trigger the initiation of the antibody-forming mechanism for deaggregated BGG (DBGG) or DNP-BGG through their actions on the carrier-specific T-cell function. All of these PLA tested also acted more or less to inhibit the induction of the carrier-specific T-cell tolerance to DBGG. Moreover, some of these PLA could act to augment antibody response to DNP-Ficoll. The adjuvant action of PLA in the response to DNP-Ficoll worked as well in athymic nu/nu mice as in nu/+ mice, whereas that in the response to DNP-BGG did not occur in athymic nu/nu mice. The order of the strength of the action of PLA to trigger the initiation of the whole immune response to DBGG, that to trigger the carrier-specific T-cell function to DNP-BGG, and that to inhibit the induction of the whole tolerance to DBGG was very similar to each other: i.e., CPS-K ? Con A > LPS, DS, poly A:U, PWM and PHA. By contrast, the order of the strength of the action to inhibit the induction of T-cell tolerance to DBGG was ≧ = LPS > Con A, PWM and poly A:U > DS and PHA, and that of the action to augment the antibody response to DNP-Ficoll was CPS-K > LPS > Con A. CPS-K was the most potent in all of these immunological activities. It was concluded that PLA act generally to stimulate the immune response at its initiation step in which T cells in the case of T-dependent antigen and B cells in the case of T-independent antigen play a predominant role, but that individual PLA share this adjuvant activity in different fashions.     

Potentiation and modulation of the immune response of guinea pigs to poorly immunogenic protein-hapten conjugates by pretreatment with the MER fraction of attenuated tubercle bacilli

The MER fraction of attenuated tubercle bacilli of the BCG strain was shown capable of stimulating and modulating the immunological responsiveness of guinea pigs to immunization with DNP conjugates of allogeneic globulin (DNP-GPG) and xenogeneic albumin (DNP-HSA). These antigens are very poorly immunogenic and fail to evoke detectable immune responses following single administration alone.When incorporated in incomplete Freund's adjuvant (IFA) together with the conjugates, MER could substitute for whole tubercle bacilli in the adjuvant mixture, and cause the conjugates to evoke both cellular and humoral reactivity, the former indicated by the development of skin reactions of delayed type (DH) to test injections of the antigens, the latter by the formation of humoral antibodies detected by an indirect hemagglutination (HA) test. When administered in saline together with antigen, MER was ineffective.Pretreatment with MER by any of several different routes 7 or 14 days prior to sensitization enabled a large number of the animals to respond with either DH, circulating antibody formation, or both. Under similar circumstances, pretreatment with Freund's complete adjuvant (FCA) elicited no such preparatory effect. In order to be efficacious in pretreatment, MER had to be given in a saline suspension; activity was lost when it was applied in IFA.MER pretreatment modulated the immune response to subsequent sensitization with the conjugates preferentially towards DH or antibody production, depending on the parameters of treatment and specific immunization. It appeared that when the specific immunogenic stimulus was weak, pretreatment with MER strongly favored DH.     

Effects of recombinant cholera toxin b subunit (rCTB) on cellular immune responses: enhancement of delayed-type hypersensitivity following intranasal co-administration of Mycobacterium bovis-BCG with rCTB

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. To gain insight into the mechanism underlying the adjuvant effect of rCTB, the effects of rCTB on cell-mediated immune responses of mice and guinea pigs were examined after intranasal administration of Mycobacterium bovis -bacillus Calmette-Guérin (BCG) with and without rCTB. Delayed-type hypersensitivity, for skin reactions in guinea pigs and for footpad swelling reactions in mice, to purified protein derivative (PPD) were enhanced by intranasal co-administration of BCG and rCTB, as compared to giving BCG alone to these animals. Moreover, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma production of spleen cells and antigen specific spleen cell proliferation, stimulated with PPD, were enhanced in the presence of rCTB. These results strongly suggest that rCTB enhances cellular as well as humoral immune responses.     

Alveolar macrophages in pulmonary immune responses. I. Role in the initiation of primary immune responses and in the selective recruitment of T lymphocytes to the lung

Antigen inoculated intratracheally (IT) into animals can induce primary immune responses and selectively recruit specific T cells to the lung. In the current study, the role of alveolar macrophages (AM) in these two responses was investigated. Antigen-pulsed bronchoalveolar cells (BAC) inoculated IT into guinea pigs generated a population of immune T cells that proliferated in vitro on reexposure to antigen-pulsed macrophages (M?). The possibility that antigen-pulsed donor BAC shed antigen that was subsequently processed and presented by host M? was ruled out by genetic experiments. Thus, peritoneal exudate lymphocytes (PEL) from (2 X 13)F1 guinea pigs primed with antigen-pulsed BAC from strain 2 animals responded preferentially to antigen-pulsed strain 2 M? rather than to antigen-pulsed strain 13 M?. In a second set of studies, antigen-pulsed BAC inoculated IT into guinea pigs selectively recruited antigen-specific T cells to the lung. Genetic experiments verified that inoculated BAC were the source of the antigen-presenting cells responsible for selective recruitment. Thus, antigen-pulsed strain 2 BAC inoculated IT recruited a greater proportion of (2 X 13)F1 T cells that recognized antigen in the context of strain 2 M? than F1 T cells that recognized antigen on strain 13 M?. Taken together, these studies suggest that AM contribute to the regulation of pulmonary immunity by both inducing T lymphocyte immunity and selectively recruiting specific T cells to the lung.     

Protective immune response to 16 kDa immunoreactive recombinant protein encoding the C-terminal VP1 portion of Foot and Mouth Disease Virus type Asia 1.

Recombinant protein of Foot and Mouth Disease Virus (FMDV) type Asia 1 corresponding to the C-terminal half of VP1 was expressed in Escherichia coli. As an alternative to the synthetic peptide, this selected C-terminal region was used as a protein vaccine in guinea pigs in order to study the immune response with various adjuvant formulations: immune stimulatory complexes (ISCOMs), Montanide ISA 206, Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS) and cytokine mixture. A primary dose of 40 microg/animal followed by a booster of the same dose was injected after a 21-day interval. The sera were collected at intervals of 21, 42 and 63 days after the booster. The humoral response to vaccine was monitored by sandwich enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test (SNT). The guinea pig sera showed high titers both in ELISA and SNT, which could be protective. Further, irrespective of the adjuvant preparation used, the vaccine conferred protection against the challenge virus 105 days post-vaccination in 13 of 15 animals (86%). The results indicated that a combination of recombinant protein ISCOMs and Montanide ISA 206 would be a better choice for achieving early protective titers and longer lasting immunity and that the C-terminal half of the VP1 protein may be tried as a safe vaccine for secondary immunization.     

Experimental cryptococcosis in guinea pigs

A guinea pig model was used to evaluate immune response to Cryptococcus neoformans. This model shared characteristics with cryptococcosis in humans. Twenty five guinea pigs injected intraperitoneally with 10(7) viable C. neoformans cells developed disseminated disease. Forty days after infection all guinea pigs were killed and autopsy performed. C. neoformans growth in the lungs, brains, livers and spleen of the infected animals were determined. Furthermore, the immune response was characterized by moderate degree of delayed-type hypersensitivity and humoral response. In some organs was observed neutrophil and lymphocyte infiltration with presence of cryptococci cells. The infiltration observed in the organs was probably a consequence of an immune reaction.     

Functional, structural, and antigenic similarities of guinea pig anti-phosphorylcholine antibodies.

The humoral immune response to PC was measured in guinea pigs. PC-vaccine stimulated IgM and IgG2, but little IgG1, anti-PC -antibodies. No memory was induced and immunization in CFA produced tolerance. PC-KLH, on the other hand, stimulated IgM, IgG2, and IgG1 anti-PC antibodies with carrier-specific memory. Hapten inhibition of plaque formation showed uniform binding patterns with minor, but significant, differences between antiPC-vaccine and anti-PC-KLH antibodies. The antibodies were characterized by IEF and idiotypic analyses. Early after immunization with PC-vaccine, guinea pigs had restricted IEF patterns which in inbred, but not outbred animals were indistinguishable between individuals. These patterns remained restricted but more individualized with time after immunization. Anti-PC-KLH antibodies showed more heterogeneity and individuality. However, these structurally heterogeneous antibodies reacted equivalently with rabbit anti-idiotype antisera and therefore must share common structural features, regardless of isotype or the genetic background of the guinea pig.     

Does immune challenge affect torpor duration?

1. Hibernation may alter the relationship between pathogens and their hosts; low host temperatures can prevent pathogen replication. Therefore, manipulating the timing and duration of torpor bouts could allow animals to gain an advantage over pathogens.
2. Thirty-two Turkish Hamsters ( Mesocricetus brandti ) were placed in short-day, cold conditions. After 10 weeks, 20 animals were challenged with an antigen to simulate a pathogen infection. Ten of these animals were returned to the cold ('cold-challenged'). The other 10 animals were placed in warm conditions ('warm-challenged'). Twelve animals received saline injections and were returned to the cold ('cold-control'). Cold-challenged animals spent significantly more time in torpor than did cold-control animals.
3. After 6·5 weeks, all animals were housed in warm conditions and ceased torpor. Both cold-challenged and warm-challenged animals received a second injection of antigen. There was no correlation between time spent euthermic and level of secondary humoral response of cold-challenged animals. The secondary humoral response of the cold-challenged animals was significantly lower than that of warm-challenged animals.
4. In this study immune status influenced torpor duration, and torpor caused immunosuppression. Hibernators may manipulate body temperature in order to combat pathogens while their own immune systems are suppressed.     

Decreased anaphylactogenic properties of disaggregated gamma-globulin]

Immunological properties of native, aggregated, and disaggregated (tolerogenic) human gamma-globulin were compared in experiments on mice and guinea pigs. Disaggregated preparation was shown to give diminished local and general hypersensitivity reaction in sensitized animals and had decreased ability to induce secondary humoral immune response.     

Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole.

Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.